Many inherited diseases are the result of a single difference in the genetic code for a particular protein. As a result of that difference, either a protein is not made at all, made in inadequate amounts, or made in a defective form. The disease is therefore a result of a person either not making a protein or not making enough of a protein, or having a defective form of it.
In the early 1990's, researchers identified a new type of mutation called dynamic or expansion mutations. Researchers had noted that in a variety of diseases, there was an increase in severity of a disease or earlier onset of a disease over several generations. Today we understand these diseases to be trinucleotide repeat sequence disorders.
A trinucleotide repeat sequence disorder, also known as a trinucleotide repeat disorder, a trinucleotide repeat expansion disorder or a triplet repeat expansion disorder, is a genetic disorder caused by an increase in the number of trinucleotide repeats in a, gene exceeding a normal, stable, threshold.
Trinucleotide repeat sequence disorders are divided into two categories determined by the type of repeat. The most common repeat is a repeat of the triplet nucleotide sequence CAG which, when present in the coding region of gene, codes for the amino acid glutamine (Q). Therefore, these disorders are referred to as polyglutamine (polyQ) disorders. Disorders that not involve a repeat of the CAG triplet nucleotide sequence, or in which a CAG triplet nucleotide sequence is not in the coding region of the gene are referred to as non-polyglutamine disorders.
Fragile X syndrome (FXS) is the most common inherited mental retardation disorder, which results in a spectrum of physical and intellectual limitations and emotional and behavioral features which range from severe to mild in manifestation. FXS is also the most common known cause of autism or “autistic-like” behaviors which can include its characteristic physical and behavioral features as well as delays in speech and language development.
The X-linked Fragile X Mental Retardation 1. (FMR1) gene is responsible for FXS. Most FMR1 gene mutations involve expansions of a polymorphic stretch of CGG repeats in its 5′ untranslated region. Unaffected individuals carry alleles ranging from 6 to 44 repeats, which are stably transmitted from generation to generation. Individuals with FXS carry full mutation alleles with >200 repeats which is accompanied by hypermethylation of the FMR1 promoter region and gene silencing. Alleles with 45 to 54 repeats are classified as gray zone alleles. Although gray zone alleles are associated with some degree of size instability, they are more stable than premutation alleles, which range from 55 to ˜200 repeats. Premutation alleles are meiotically unstable and may expand from one generation to the next. These alleles have also been associated with high transcript but low peptide levels and may be linked to disorders that are clinically distinct from FXS, such as fragile X-associated tremor ataxia syndrome (FXTAS) in males and premature ovarian failure (POF) in females.
The FMR1 CGG repeat is normally interspersed by AGG trinucleotide interruptions after every 9 or 10 CGGs, with most normal alleles containing two AGG interruptions and most premutation alleles containing only one AGG interruption at the 5′ end of the repeat region or none at all. The loss of an AGG interruption, especially at the 3′ end of a repeat region, results in a long stretch of uninterrupted CGG repeats that has been associated with CGG repeat instability, especially in alleles with >24 uninterrupted CGG repeats at the 3′ end of the repeat.
The FMR1 gene is located on the X chromosome. Therefore, since a female has two X chromosomes, a female with a premutation or full mutation has a 50% chance of passing on the X with the mutation in each pregnancy. If she has a premutation, this can be passed onto her offspring where it can either remain as a premutation or it can expand to a full mutation. Unlike many other X-linked conditions where only males who inherit the abnormal gene are affected (since they only have one X chromosome and do not have another normal copy to compensate), females can also be affected by FXS.
At this time, there is no, cure for FXS. Currently, the syndrome is treated through a combination of behavioral therapy, special education, and when necessary, treatment of physical abnormalities. Persons with relatives suffering from FXS are advised to seek genetic counseling to assess the likelihood of having children who are affected, and how severe any impairments may be in affected descendants. This is especially important because individuals who carry the premutation alleles are non-symptomatic and it may not be readily apparent that such an individual is at risk of having offspring with FXS.
The most commonly used method of diagnosis of FXS is DNA testing by PCR amplification across the triplet repeat stretch, supplemented by Southern blot analysis. While PCR analysis is able to size all normal and gray zone alleles, as well as small premutation alleles, larger expansions are refractory to PCR amplification due to their large amplicon sizes and high GC contents. Conventional PCR based approaches are also unable to provide information on the methylation states of the repeats.
Conversely, Southern blot analysis can detect large expansion mutations but cannot accurately distinguish large normal or gray zone alleles from small premutation alleles, requires large amounts of DNA and is also highly labour-intensive. Currently, a combination of both methods is necessary to ensure accurate FMR1 CGG repeat classification, making molecular diagnosis and screening of suspected FXS patients time-consuming and costly.
To get around this problem, several methylation-specific PCR methods have been developed to detect FMR1 CGG repeat expansions, taking advantage of sequence variations between methylated and unmethylated DNA after treatment with sodium bisulfite to assist in discrimination between normal and expanded alleles. Sodium bisulfite treated DNA is also less GC rich and easier to amplify. However, these assays often involve multiple PCR reactions, or can only be used for analysis of male samples. Also, since FMR1 resides on the X chromosome, interpretation of assay results of females are often complicated by the presence of two X chromosomes, one of which will be inactivated for X-linked gene dosage compensation.
Presently, the absence of a quick and robust genetic screen for FXS and its related syndromes is one of the main limiting factors to the implementation of a routine genetic screen of the FMR1 locus. Hence, there is an urgent need for improved methods for screening for FXS.
There is a need to provide a fast reliable method that overcomes, or at least ameliorates, one or more of the disadvantages described above.